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Home , Arthrobotrys, Arthrobotrys dactyloides, Arthrobotrys oligospora, Aspergillus nidulans, Dactylellina, Monacrosporium

Natural diversity in the predatory behavior facilitates the establishment of a robust model strain for -trapping fungi

Ching-Ting Yanga,1, Guillermo Vidal-Diez de Ulzurruna,1, A. Pedro Gonçalvesa, Hung-Che Lina,b, Ching-Wen Changa,c, Tsung-Yu Huanga, Sheng-An Chena, Cheng-Kuo Laid, Isheng J. Tsaid, Frank C. Schroedere,f, Jason E. Stajichg, and Yen-Ping Hsueha,b,c,2

aInstitute of Molecular Biology, Academia Sinica, Nangang, Taipei 115, Taiwan; bGenome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 106, Taiwan; cDepartment of Biochemical Science and Technology, National Taiwan University, Taipei 106, Taiwan; dBiodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan; eBoyce Thompson Institute, Cornell University, Ithaca, NY 14853; fDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; and gDepartment of Microbiology and Plant Pathology, University of California, Riverside, CA 92521

Edited by Iva Greenwald, Columbia University, New York, NY, and approved February 5, 2020 (received for review November 9, 2019) Nematode-trapping fungi (NTF) are a group of specialized microbial the induction of trap morphogenesis (7), and that NTF use predators that consume when food sources are limited. olfactory mimicry to lure their prey into traps (8). Upon being Predation is initiated when conserved nematode ascaroside pher- trapped, the nematodes are paralyzed, pierced, invaded, and omones are sensed, followed by the development of complex digested by the (5, 6). Both nematodes and NTF can be trapping devices. To gain insights into the coevolution of this found in a wide range of habitats (9, 10); however, sympatrism – interkingdom predator prey relationship, we investigated natural and adaptation in the context of their relationship are yet to be populations of nematodes and NTF that we found to be ubiqui- investigated and little is known about the natural history of this tous in soils. were sympatric with various example of a predator–prey interaction. nematode species and behaved as generalist predators. The ability In this study, we investigated the ecology of natural populations to sense prey among wild isolates of var- of nematodes and NTF and found that Arthrobotrys species were ied greatly, as determined by the number of traps after exposure sympatric with diverse nematodes and behaved as generalist MICROBIOLOGY to elegans. While some strains were highly sensi- tive to C. elegans and the nematode pheromone ascarosides, others responded only weakly. Furthermore, strains that were Significance highly sensitive to the nematode prey also developed traps faster. The polymorphic nature of trap formation correlated with compe- Nematode-trapping fungi (NTF) are carnivorous microbes that tency in prey killing, as well as with the phylogeny of A. oligospora hold potential to be used as biological control agents due to natural strains, calculated after assembly and annotation of the their ability to consume nematodes. In this work, we show that of 20 isolates. A -level assembly NTF are ubiquitous generalist predators found in sympatry and annotation were established for one of the most sensitive with their prey in soil samples. Wild isolates of NTF displayed a wild isolates, and deletion of the only G- β-subunit– naturally diverse ability to execute their predatory lifestyle. We encoding of A. oligospora nearly abolished trap formation. generated a large whole-genome sequencing dataset for many In summary, our study establishes a highly responsive A. oligospora of the fungal isolates that will serve as the basis of future wild isolate as a model strain for the study of fungus–nematode projects. In particular, we establish TWF154, a highly respon- interactions and demonstrates that trap formation is a fitness sive strain of Arthrobotrys oligospora, as a model strain to character in generalist predators of the nematode-trapping fungus study the of NTF. Lastly, we provide evidence that the family. G-protein β-subunit Gpb1 plays an important role in trap in- duction in NTF. nematode-trapping fungi | predator–prey interaction | natural population | G-protein signaling | trap morphogenesis Author contributions: C.-T.Y., G.V.-D.d.U., and Y.-P.H. designed research; C.-T.Y., G.V.-D.d.U., H.-C.L., C.-W.C., T.-Y.H., S.-A.C., and Y.-P.H. performed research; H.-C.L., C.-W.C., S.-A.C., F.C.S., J.E.S., and Y.-P.H. contributed new reagents/analytic tools; C.-T.Y., G.V.-D.d.U., A.P.G., H.-C.L., he living components of soils are in permanent interaction C.-W.C., T.-Y.H., C.-K.L., I.J.T., and Y.-P.H. analyzed data; and G.V.-D.d.U., A.P.G., S.-A.C., and Tand play central roles in various aspects of biogeochemistry, Y.-P.H. wrote the paper. including nutrient cycling and transport across distances (1). The authors declare no competing interest. Specifically, nematodes are the most abundant in nature, This article is a PNAS Direct Submission. 20 amounting to a staggering 0.3 gigatonnes or 4.5 × 10 individ- Published under the PNAS license. uals, many of which are devastating parasites of plants, animals, Data deposition: The genomic data presented in this paper have been deposited to and humans (2). Although available, nematicide chemicals pose National Center for Biotechnology Information GenBank. The accession numbers are severe environmental and health risks and therefore biological SOZJ00000000 (TWF154 reference genome), MN972613 (TWF154 mitochondria), WIQW00000000 (TWF102), WIQX00000000 (TWF103), WIWS00000000 (TWF106), control agents such as carnivorous predatory fungi hold potential JAABOH000000000 (TWF128), JAABLO000000000 (TWF132), JAAGKP000000000 (TWF173), as alternative pest control tools. Predatory fungi that prey and WIPF00000000 (TWF191), WIPG00000000 (TWF192), JAABOI000000000 (TWF217), consume nematodes have been described from several major WIWR00000000 (TWF225), WIRA00000000 (TWF569), WIRB00000000 (TWF594), WIWT00000000 (TWF679), WIQZ00000000 (TWF703), WIQY00000000 (TWF706), fungal phyla, suggesting that fungal carnivorism has arisen in- WIWQ00000000 (TWF751), JAABOE000000000 (TWF788), and JAABOJ000000000 dependently multiple times during evolution (3, 4). In partic- (TWF970). References to these accession numbers can also be found throughout this paper ular, nematode-trapping fungi (NTF) constitute a group of and in SI Appendix. nematophagous organisms that includes a large number of species 1C.-T.Y. and G.V.-D.d.U. contributed equally to this work. that rely on the formation of traps to ambush their prey (5, 6). We 2To whom correspondence may be addressed. Email: [email protected]. have previously described that NTF eavesdrop on an evolutionarily This article contains supporting information online at https://www.pnas.org/lookup/suppl/ conserved family of nematode pheromones, the ascarosides, that doi:10.1073/pnas.1919726117/-/DCSupplemental. are used as a signal to switch from saprophytism to predation via

www.pnas.org/cgi/doi/10.1073/pnas.1919726117 PNAS Latest Articles | 1of9 Downloaded by guest on September 24, 2021 predators. The degree of predation varied greatly among strains vast majority of the 178 soil samples and ∼92 and ∼68% of the and several wild isolates were substantially more efficient pred- sampling sites contained nematodes and/or NTF, respectively. ators than the currently used laboratory strain. We sequenced They were sympatric in more than 63% of the sites, indicating that and assembled the genomes of a number of wild Arthrobotrys these organisms share the same niches in soil environments and oligospora strains and demonstrated a correlation between the interact closely in nature (Fig. 1B). Only 2 sampling sites (the levels of trap formation, prey killing efficiency, and phylogeny. Xiaoyoukeng fumarole at Yangmingshan National Park and the Lastly, targeted gene deletion of a vG-protein β-subunit–encoding coastline of Penghu Island) were found to be free of nematodes gene revealed that the predatory lifestyle of A. oligospora is de- and NTF, presumably due to extreme environments (high tem- pendent upon G-protein signaling. perature and high salt concentration, respectively) that likely impede survival. In total, we identified 16 previously described Results and Discussion species of NTF, as well as a few strains that could represent new Soil Samples Reveal a Rich Diversity of Nematodes and NTF. With the species. Among the 16 known species, 11 were identified as goal of isolating sympatric nematodes and NTF from natural belonging to the Arthrobotrys, which develops 3D adhesive habitats, we collected 178 soil samples from 69 ecologically diverse nets as a trapping device to capture prey (5, 6) (Fig. 1C). A. sites in Taiwan (Fig. 1A). Sampling sites were mainly located in oligospora was the most commonly found species, followed by northern and central Taiwan, and included forest, lake, agricul- Arthrobotrys thaumasia and Arthrobotrys musiformis; combined, tural, and coastal environments as well as educational campuses. these 3 species were present in ∼70% of the sampling sites that More than 66% of the sampling sites were mountainous areas, had been identified as harboring NTF (Fig. 1C). The remaining with altitudes ranging from ∼300 to 3,400 m (SI Appendix,Table 5 species were of the and Drechslerella genera, S3). We established pure cultures of both organisms and their which develop adhesive columns and constricting rings as trap- identities were confirmed by sequencing the internal transcribed ping devices (4), and were each isolated from 1 sampling site spacer (ITS) region of the fungal strains and the small-subunit only (Fig. 1C). Approximately 35% of the sampling sites con- (SSU) ribosomal DNA (rDNA) regions of the nematodes. By tained 2 or more species of NTF, indicating that different species not applying a metagenomic sequencing approach, we have likely of NTF can occupy the same ecological niches (SI Appendix, underestimated the fungal and nematode biodiversity in our Table S3). Conidial morphology was diverse among the isolated samples. Nevertheless, our approach enabled us to establish cul- NTF (Fig. 1C). tivable isolates of NTF and nematodes and examine their inter- Nematodes were present in ∼93% of the soil samples (Fig. actions in the laboratory. We identified nematodes and NTF in the 1B). We cultured the wild nematodes on standard Caenorhabditis

AC

0.050

BD40

Neither Only nematodes 30 3% 29% Cephalobidae Only nematm 29% 20

10 Only NTF 4% Both 0 64% ) ) ) ) ) ) (5) (1) p. s sp. (1) ti cus sp. (1) chus sp. (4) Zeldia sp. (1) Alaimus sp. (1 Plectusion sp. (2) OscheiusPelodera s sp. (3)Rhabditis toplectussp. (5) sp. (1) cephalus sp. Cervidellus sp. (2) Bunonema sp. (2 Rhabditella sp.(1) Demaniella sp. (1) ist Wilsonema sp. (2 CephalobusChiloplacus sp. (1) sp.Acrostichus (1) sp. (1) DiploscapterEcumeni sp.Mylonchulus (1) Panagrellus sp. (1) sp.Pr (2) Acrobeloides sp. (6 Aphelenchus sp. (1) Mesorhabditis Protorhabditissp. (2 sp. (1)Cera Cephaloboides sp. (1) CaenorhabditisDiplogastrellus sp. (5) sp. (1) Choriorhabdi EumonhysteraPanagrolaimus sp. (1) sp. (4) Pseudacrobeles sp. (1) AphelenchoidesAporcelaimellus sp. (3) sp. (2) Rhabditophanes sp. (1) Terato Bursaphelenchus sp. (2 Diplogasteroides sp. (1) Sheraphelenchus sp. (1)

Fig. 1. High diversity of nematodes and nematode-trapping fungus species are sympatric in soil samples. (A) Geographic distribution of the sampling sites in Taiwan. (B) Nematodes and NTF are sympatric in more than 60% of our sampling sites. (C) Phylogeny of the different species of NTF isolated in this study based on their ITS sequences. The numbers represent the prevalence of the difference species of NTF in soil samples. Trap types and images of conidia are shown (Right). AC, adhesive columns; AN, adhesive networks; AK, adhesive knobs; CR, constricting rings. (D) Prevalence and diversity of nematode species in Taiwanese soil samples.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1919726117 Yang et al. Downloaded by guest on September 24, 2021 elegans nematode growth media (NGM) with Escherichia coli A (OP50) as food source, and directly amplified and sequenced the SSU rDNA regions from those able to grow on OP50. We identified more than 74 Nematoda species (Fig. 1D), covering clades II, IV, and V (SI Appendix, Fig. S1), demonstrating a rich nematode biodiversity in Taiwanese soil environments. tipulae, Acrobeloides apiculatus, and Acrobeloides nanus were found in ∼40, ∼30, and ∼9% of the sampling sites, respectively (Fig. 1D), in line with previous surveys that showed that these nematodes, especially O. tipulae, are widespread cosmopolitan soil nematodes (11–15).

Arthrobotrys Fungi Behave as Generalist Predators. A. oligospora, A. thaumasia, and A. musiformis, the 3 most commonly identified NTF in our soil samples, were sympatric with at least 50, 29, and 31 species of nematodes, respectively, indicating that NTF natu- rally encounter a broad range of nematodes, including several Caenorhabditis species (SI Appendix,TableS4). C. elegans is known to be associated with rotten fruits (16), and in 1 soil sample col- lected from an apple orchard, we identified both C. elegans and A. oligospora, providing direct evidence that C. elegans en- counters A. oligospora in its natural habitat. To assess nema- tode predation, we tested the trapping ability of A. oligospora, A. thaumasia,andA. musiformis on sympatric nematode species and observed that all 3 Arthrobotrys preyed on all of the nema- tode species for which we were able to establish cultures (Fig. 2A). Importantly, when nematodes and NTF from distant sam- pling sites were cocultured, trap formation and carnivorism were MICROBIOLOGY still observed. For example, the nematodes Pristionchus pacificus and Cervidellus vexilliger were not sympatric with A. oligospora in any of the samples, yet A. oligospora still developed traps and consumed isolates of these 2 nematode species (Fig. 2B). Thus, we conclude that A. oligospora does not specifically recognize and prey on particular species of nematodes but instead behaves as a generalist predator. These data support previous findings that highly conserved nematode signals such as ascarosides play a critical role in this predator–prey interaction (7). Additionally, our observation that multiple nematode-trapping fungus species coexist in the same sample suggests that they might compete for prey in their natural environments. B Predation Is a Highly Polymorphic Trait in Arthrobotrys Species. Phenotypic diversity is commonly observed in natural populations of fungi. For example, variations in stress sensitivity have been observed in (17) and (18). Two previous studies have surveyed A. oligospora in the wild (19, 20) but no phenotypic characterization has been per- formed. Thus, we designed an assay to quantitatively measure the ability of individual isolates to sense prey and develop traps, the most distinctive feature of NTF, and examined the natural diversity of nematode-trapping performance. Wild NTF were exposed to 30 C. elegans L4 larvae for 6 h and the number of traps developed in cultures at 24 h postexposure to nematodes was determined. The average prey-sensing ability of 35 of the A. oligospora wild isolates was highly polymorphic for this particular trait, ranging from ∼8 to 162 traps (Fig. 3A; raw data are available in SI Appendix,TableS5). It is noteworthy that even TWF788, the lowest-performing isolate from our samples, formed, on average, a Fig. 2. A. oligospora, A. musiformis,andA. thaumasia are generalist preda- tors. (A) NTF preying on different sympatric nematode species. (Scale bar, higher number of traps than the most commonly studied labora- 200 μm.) (B) NTF preying on allopatric nematode species. tory strain of A. oligospora, ATCC24927 (21), while the highest- performing strains, TWF132 and TWF154 (SI Appendix,Fig.S2), exhibited almost 20-fold more traps than ATCC24927. In fact, our quantitative approach revealed several statistically different groups While some wild isolates were highly responsive, others were and at least 21 wild isolates outperformed ATCC24927. A poly- almost insensitive to this cue, and ATCC24927 was again among the morphic response that ranged from ∼8to92trapswasalsoob- lowest-performing statistical group (Fig. 3B). This finding demon- served when fungal hyphae were exposed to synthesized ascaroside strates that ascaroside-triggered trap morphogenesis is highly poly- #18 (Fig. 3B; raw data are available in SI Appendix, Table S6). morphic among our A. oligospora wild isolates. Responsiveness to C.

Yang et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 24, 2021 A

B

CDG

E

F

Fig. 3. Prey-sensing ability varies considerably among wild isolates of A. oligospora.(A) Quantification of trap number induced by C. elegans WT strain N2 among wild isolates of A. oligospora (mean ± SEM, n = 6; different letters represent significant differences from Tukey test). (B) Quantification of trap number induced by ascarosides among wild isolates of A. oligospora (mean ± SEM, n = 6; different letters represent significant differences from Tukey test). (C) Quantification of trap number induced by C. elegans in wild isolates of A. musiformis and A. thaumasia (mean ± SEM, n = 6). (D) Time of trap emergence after exposition to C. elegans among different A. oligospora wild isolates. (Mean ± SEM; n = 4; different letters represent significant differences from Tukey test.) (E) Percentage of live C. elegans after exposition to different strains of A. oligospora over 12 h. (Mean ± SEM.) (F) Competition fitness assay of A. oligospora isolates TWF154 and TWF106, computed as the percentage of conidia produced by TWF154 against the total number of conidia in the presence and absence of C. elegans (N2). (Mean ± SEM; n = 7; asterisks represent significance levels of unpaired t test.) (G) Phylogeny of 19 A. oligospora wild isolates sequenced and assembled for this study. The phylogenetic tree was constructed using 500 random single orthologs and the heatmap summarizes the trapping response of the isolates toward N2 and ascarosides.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1919726117 Yang et al. Downloaded by guest on September 24, 2021 elegans was positively correlated with responsiveness to ascarosides compared with the weak predatory strain; ∼62% of the conidia (SI Appendix,Fig.S3). This correlation, although robust (Spearman, harvested from the competition assay were hygromycin-resistant r = 0.7556, P < 0.0001), was not observed throughout all of the (Fig. 3F). In contrast, in the presence of nematode prey, the fitness strains, suggesting that prey sensing is a multifactorial process. In advantage was further increased by ∼15%, since 78% of the addition, we studied the amount of phenotypic variation due to conidia harvested from the competition assay were produced by genetic variation by means of the SNP heritability in the re- the strong predatory strain (Fig. 3F). These results suggest that sponse of A. oligospora to nematode cues using the genomes of a higher predation ability results in better fitness in the nematode- subpopulation of 18 wild isolates (SI Appendix). We obtained trapping fungus A. oligospora. preliminary values of 0.473 and 0.463 for the response to nem- atodes and ascarosides, respectively, suggesting that genetic The Nuclear and Mitochondrial Genome of a Highly Responsive A. differences influenced the response of A. oligospora to prey sig- oligospora Isolate. In order to generate a genomic resource for nals. We did not observe a clear pattern when the association future studies on the molecular basis of trap morphogenesis in between geographic location and prey-sensing phenotypes was NTF, we assembled the genomes of 19 A. oligospora wild isolates examined (SI Appendix, Fig. S4). We further determined trap (Fig. 3G). Interestingly, a phylogenetic analysis using 500 ran- formation in 10 A. thaumasia and 10 A. musiformis wild isolates dom from these 19 genomes (and ATCC24927) showed a in response to C. elegans (SI Appendix,Fig.S5) and found that this clustering pattern that was correlated with the levels of trap trait was also highly polymorphic in these 2 species, thus providing formation (Fig. 3G). For example, TWF751, TWF154, TWF132, evidence that prey sensing is likely highly polymorphic in natural TWF128, TWF217, and TWF225 formed a separate clade and populations of the Arthrobotrys genus and that A. thaumasia belonged to the highest-performing group, which were most sen- strains formed substantially fewer traps than A. oligospora and sitive to the nematode prey (Fig. 3A). We anticipate that fu- A. musiformis (SI Appendix, Fig. S5). We next investigated if the ture genome-wide association studies and comparative genomics polymorphic prey-sensing phenotype displayed by A. oligospora analyses with additional genomes from the natural population wild isolates was nematode species-dependent. We selected 3 will disclose multiple candidate genetic loci that govern the A. oligospora strains that were strongly responsive (TWF132, predation trait. TWF154, and TWF145) and 4 strains that were weakly re- We decided to focus on TWF154, a high-performing wild sponsive (TWF106, TWF788, TWF191, and ATCC24927) to C. isolate, in response to C. elegans, other nematode species, and elegans and measured their ability to form traps in response to 4 ascarosides (Fig. 3 A–C), that we intend to establish as the pre- other nematode species (Caenorhabditis briggsae, O. tipulae, ferred A. oligospora model wild-type (WT) strain for further mo- Rhabditis rainai, and P. pacificus). Overall, the levels of trap lecular mechanistic studies. We used the PacBio RSII platform to MICROBIOLOGY formation were diverse among these strains and if responsiveness obtain high-quality long reads from strain TWF154 with 131× to C. elegans was higher, the sensitivity to other nematode spe- coverage and assembled a complete and contiguous genome of cies was similarly higher and vice versa (Fig. 3C). Thus, natural 39.6Mbpdistributedover10scaffolds(Fig.4A). Eight out of 10 diversity in the quantities of trap formation is not restricted to contigs represented a telomere-to-telomere complete chromo- just a single species of NTF nor is it restricted to the response to some (Chr) assembly (Chr1 to Chr8; Fig. 4A). The remaining 2 a single species of nematode. contigs (Chr9a and Chr9b) reflect 2 telomere-to-centromere We next assessed whether the onset of trap morphogenesis assemblies based on the telomeric repeat sequences identi- and the ability to kill nematode prey varied among the natural fied at one end and the transposable element clusters at the population of A. oligospora in addition to the variation in trap other consisting predominantly of long terminal repeat retro- numbers developed in response to nematode prey. We found transposons and DNA transposons, which are typical signa- that strains TWF154, TWF128, and TWF132, which formed tures of centromeres in fungi (22), suggesting that Chr9a and more traps in response to nematode prey (Fig. 3A), developed Chr9b represent 2 arms of the same chromosome (Fig. 4A). the initial traps as soon as ∼2.5 h after exposure to C. elegans Genes were distributed evenly along the with an whereas strains forming fewer traps (TWF106, TWF192, and average of 247 genes per Mbp, except for regions of low gene ATCC24927) exhibited slower onset of trap morphogenesis distribution at the centromeres (Fig. 4A). Predicted gene func- (∼5to∼7.5 h) (Fig. 3D). In line with these results, strains that tion cataloging using the cluster of orthologous groups database developed traps faster and greater in number captured the showed the presence of all major categories, although the largest nematode prey significantly faster (Fig. 3E). In our killing assays, group of genes was classified as “function unknown” (Fig. 4B). only ∼20% of nematodes were still alive 12 h after being added to The quality of the A. oligospora TWF154 genome sequence is the fungal cultures in the presence of highly responsive wild among the most contiguous and complete of the sequenced as- isolates in contrast to ∼75 to 85% survival in the case of the comycetes. General features of the genome assemblies for the A. weakly responsive wild isolates (Fig. 3E). Altogether, these re- oligospora wild isolates TWF154 and ATCC24927 (23) are pre- sults indicate that predation on nematodes is a naturally diverse sented in Table 1. For example, the number of scaffolds and N50 trait in populations of generalist NTF and that some strains have in our annotation of TWF154 (10 scaffolds and N50 = 5,390,931) evolved to become intrinsically more robust and efficient in were improved in comparison with ATCC24927 (215 and sensing nematodes, executing the trapping developmental pro- 2,037,373, respectively). Using RNA-seq data from 5 different gram, and consuming prey. Hence, trap morphogenesis seems to culture conditions as guidance and the proteomes of represen- function as a fitness characteristic in NTF. tative fungi (SI Appendix), the program Funannotate predicted 12,107 gene models in our improved A. oligospora genome as- Competition Assays Link Predation Ability to Fitness in A. oligospora. sembly; in comparison, 11,479 genes had previously been found After consuming the nematode prey, A. oligospora develop for ATCC24927 (Table 1). The proteome is 97.7% complete chains of conidia (asexual spores) (SI Appendix, Fig. S6). To test based on the BUSCO (24) assessment using the the hypothesis that predation ability contributes to the fitness of dataset, which is comparable to other published reference fungal A. oligospora, measured by the number of conidia produced, we genomes. Furthermore, we also assembled and annotated a genetically marked the strong predator TWF154 with a hygromycin- complete mitochondrial genome (Fig. 4C). Notably, the size resistant marker (TWF154-HYGR) and competed this strain of the mitochondrial genome of TWF154 (∼161 kb) is much with the weak predator TWF106 in the presence or absence of larger than that of the human (∼17 kb), N. crassa (∼65 kb), nematode prey. We found that in the absence of nematode prey, or S. cerevisiae (∼79 kb) mitochondrial genomes. The expan- the strong predatory strain exhibited a moderate fitness advantage sion of the mitochondrial genome of TWF154 and other fungal

Yang et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 24, 2021 A B

TWF154, mitochondrial 160,951 bp

C Z. Cytoskeleton (118) Y. Nuclear structure (22) W. Extracellular structures (10) B. structure and dynamics (87) V. Defense mechanisms (38) C. Energy production and conversion (268) U. Intracellular tra cking, secretion, and vesicular transport (382) D. control, cell division, chromosome partitioning (149) T. Signal transduction mechanisms (328) E. Amino acid transport and (284) F. Nucleotide transport and metabolism (122)

G. Carbohydrate transport and metabolism (516)

H. Coenzyme transport and metabolism (113)

I. Lipid transport and metabolism (258)

S. Function unknown (2014) J. Translation, ribosomal structure and biogenesis (363)

K. Transcription (330)

L. Replication, recombination and repair (219)

Q. Secondary metabolites biosynthesis, M. /membrane/envelope biogenesis (115) transport and catabolism (215) N. Cell motility (2) P. Inorganic ion transport and metabolism (180)

Fig. 4. G-protein signaling is required for prey sensing in A. oligospora.(A) Genome architecture of A. oligospora strain TWF154. Tracks (outer to inner) represent the distribution of genomic features: 1) positions (in Mb) of the 10 TWF154 contigs, with numbers indicating the order of scaffold size; 2) (along a 100-kb sliding window); 3) distribution of transposable elements (along a 10-kb sliding window); 4) telomere repeat frequency (along a 1-kb sliding window), showing that 8 contigs have 2 telomeric ends; and 5), A. oligospora species-specific gene content (along a 100-kb sliding window). (B) Predicted function of genes in the TWF154 genome cataloged using the cluster of orthologous groups database. (C) Circular map of the mitochondrial genome of A. oligospora TWF154. Tracks (outer to inner) show: 1) annotation of mitochondrial DNA-encoded genes: subunits of NADH dehydrogenase/complex I (yellow), cytochrome c oxidase/complex IV (red), and ATP synthase/complex V (blue); apocytochrome bCOB(green), ribo- somal protein S3 RPS3 (pink), and ribosomal RNA genes RNS and RNL (orange); 2) approximate location of LAGLI-DADG (light gray) and GIY-YIG en- donucleases (dark gray); 3) annotation of transfer RNA, where “t” stands for “tRNA” followed by the respective amino acid 1-letter code and number of copies.

mitochondrial genomes, namely in nematode-trapping fungus G-Protein Signaling Is Required for Prey Sensing in A. oligospora. We species (25–32), can be attributed to the presence of a substantial next performed functional studies of candidate genes that might number of genes encoding homing endonucleases (HEGs) of the be involved in sensing the nematodes. In C. elegans, ascarosides LAGLI-DADG and GIY-YIG families in intronic and intergenic are sensed by G protein-coupled receptors (GPCRs) (33, 34) and regions (Fig. 4C). It will be interesting to evaluate whether the in fungi, G-protein signaling and GPCRs are critical for sensing distribution of introns and HEGs varies in diverse Arthrobotrys environmental stimuli, including sex pheromones, and are re- strains and, more generally, the role of these genes during fungus– quired for virulence and morphogenesis in various fungal path- nematode interactions. ogens (35, 36). It had been suggested via chemical inhibitors that

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1919726117 Yang et al. Downloaded by guest on September 24, 2021 Table 1. Genomic features of the 2 assemblies of A. oligospora Materials and Methods genomes Isolation of Nematodes and NTF from Soil Samples. Nematodes and NTF were General features ATCC24927 TWF154 isolated using the soil sprinkle method (54) with some modifications. Briefly, soil samples were sprinkled on low-nutrient medium (LNM) and, after 2 to 7 d, Size of assembled genome, bp 40,072,829 39,619,008 nematodes were transferred to NGM containing E. coli OP50. SSU rDNA se- Number of scaffolds 215 10 quences were PCR-amplified directly from the nematodes using the single-worm N50 scaffold size, bp 2,037,373 5,390,931 PCR protocol with primers G18S4a (5′-GCTCAAGTAAAAGATTAAGCCATGC-3′) N90 scaffold size, bp 691,077 2,932,219 and DF18S-8 (5′-GTTTACGGTCAGAACTASGGCGG-3′) (55, 56). The National GC content, % 44.45 43.96 Center for Biotechnology Information (NCBI) basic local alignment search tool Number of genes 11,479 12,107 (BLAST) (57) was used to determine the identity of the nematode isolates to Number of tRNA genes 145 102 species level (if the identity was higher than or equal to 97% when compared Busco completeness, % 97.4 97.7

A G might be involved in the closure of the constriction ring of another nematode-trapping fungus, Drechslerella dactyloides (37). Furthermore, a recent study showed that the disruption of a Rab GTPase, AoRab-7A, abolished trap formation in A. oligospora (38). For these reasons, we investigated the role of G-protein signaling in prey sensing in A. oligospora. We identified a single G-protein β-subunit gene (GPB1, EYR41_009480) and 3 G-protein α-subunit genes (EYR41_010456, EYR41_011543, and EYR41_011720) in the genome of A. oligospora. Since the Gαs are known to have overlapped functions in many fungi (39), we decided to test the function of the G-protein β-subunit first. We obtained 2 independent gpb1 deletion mutants generated via homologous recombination and determined that the efficiency

of homologous recombination was extremely low (∼3%). The MICROBIOLOGY 2 gpb1 mutants were strongly defective in trap morphogenesis in response to C. elegans and ascarosides, and exhibited no obvious growth defects (Fig. 5). This phenotype was complemented by reintroducing the WT copy of the GPB1 gene (Fig. 5 A and B). These results demonstrate that G-protein signaling is required for ascaroside sensing and/or trap formation in A. oligospora and that the ascaroside receptors in A. oligospora might be GPCRs. Conclusion Our study revealed that NTF are widespread in soils of distinct ecological provenances, behave as generalist predators, are sympatric with diverse species of nematodes, and vary consid- erably in their spatial and temporal ability to capture the nem- atode prey. NTF hold great potential to be utilized as biological control agents in agricultural settings. However, only recently BC have the molecular mechanisms underlying the singular biology of these specialized predators begun to be uncovered. For ex- ample, a recent work has generated a strain lacking the signaling ** scaffold protein Soft and shown that cell–cell fusion is required for ring closure in flagrans (40). In A. oligospora, ATCC24927-background trapping-deficient have been isolated: adhesin protein Mad1 (41), autophagy protein Atg8 (42), mitogen-activated protein kinase Slt2 (43), pH sensor PalH (44), NADPH oxidase NoxA (45), low-affinity calcium uptake **** **** system proteins (46), Rab GTPase Rab7A (38), actin-associated n= 12 6 6 7 protein Crn1 (47), Woronin body component Hex1 (48), malate synthase Mls (49), glycogen phosphorylase Gph1 (50), tran- scription factors VelB (51) and StuA (52), and microRNA- processing protein Qde2 (53), to which we add the G-protein Fig. 5. G-protein β-subunit Gpb1 plays an important role in trap induction β-subunit Gpb1. However, a mechanistic view of how these in A. oligospora.(A) Images of the traps induced by C. elegans laboratory and other molecular players are interconnected during the in- strain N2 or ascarosides (ascr#7 and ascr#18) in the WT (TWF154), gpb1 teraction of A. oligospora with their prey is totally lacking. The mutants, and gpb1 GPB1 rescued strain. (Scale bar, 200 μm.) (B) Quantifi- establishment of a high-efficiency A. oligospora model strain cation of the trap numbers induced by C. elegans WT strain N2 or ascarosides (TWF154) and the generation of publicly available whole-genome (ascr#7 and ascr#18) in the WT (TWF154), gpb1 mutants, or gpb1 GPB1 reconstituted strain (n shown along the x axis). (Mean + SEM; n shown along sequencing resources for an ample number of wild isolates will the x axis; asterisks represent significance levels of unpaired t test compared pave the way to the identification of novel loci underlying phe- to the WT.) (C) Fungal colonies developed by the WT (TWF154), gpb1 mu- notypic traits and, more generally, to the fast advancement of re- tants, and gpb1 GPB1 rescued strain after 5 d of growth on PDA plates (5-cm search with NTF. diameter).

Yang et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 24, 2021 with a known species) or to the genus level (if the identity was lower than supernatant, yielding ∼0.2 mL highly concentrated spore solutions. Be- 97%). Once nematodes had been isolated from the LNM plates, C. elegans tween 8 and 12 μL of each spore suspension were added to 9-cm plates were added to the same plates to induce trap formation in the NTF. After 3 to containing PDA and PDA with 50 μL/mL HygB, respectively; all plates were 7 d, the plates were examined under a stereo microscope and single conidia treated with 90 μL 11 mg/mL ampicillin before the spore solution was of the NTF were isolated and transferred individually to potato dextrose added to prevent bacterial contamination. After 24 h, the germinated agar (PDA) to establish pure cultures. ITS regions were directly amplified spores in each plate were counted using a Zeiss Stemi 305 microscope. from fungal hyphae by PCR with the universal primers ITS1 5′-TCCGTAGGT- GAACCTGCGG-3′ and ITS4 5′-TCCTCCGCTTATTGATATGC-3′ following the TWF154 Genome Assembly and Annotation and Phylogenetic Analyses of single-worm PCR protocol (56, 58). Species identity was assigned if the ITS Nematode-Trapping Fungi. The TWF154 reference genome was sequenced sequence identity was 97% or higher from NCBI BLAST (57) analyses. Strains of and assembled from ∼0.5 million long reads with an average length of NTF and nematodes used in this study are listed in SI Appendix,TableS7.The 11,258 bp sequenced from the Pacbio RSII platform and polished with 18 SSU rDNA and ITS sequences are available in the NCBI with the accession million 250-bp Illumina reads. Annotation was done using Funannotate (65) numbers provided in SI Appendix, Table S8. C. elegans animals employed in this and the pipeline described at https://funannotate.readthedocs.io/en/latest/ study were maintained following standard procedures (55). tutorials.html. Annotation of the mitochondrial genome was performed using a combination of MFannot (66) and MITOS2 (67). Details can be found Phenotypical Characterization. To quantify trap formation, fungal isolates in SI Appendix. The neighbor-joining algorithm of MEGA7 or MEGAX was were grown on LNM for 5 d, transferred to a fresh LNM plate, and, after ∼48 h used to construct maximum-likelihood phylogenetic trees. The tree of 16 (25 °C, dark), exposed to 30 N2 C. elegans L4-stage larvae for 6 h (after which different nematode-trapping fungus species isolated from soil samples was the animals were removed with a pick) or 1 μL 1 mM ascaroside #18 (ascr#18) constructed based on ITS nucleotide sequences. Tuber melanosporum was (59) that was added ∼2 mm away from the border of hyphal tips. We fo- the outgroup. The tree of the 20 A. oligospora isolates was constructed using cused on ascr#18 because it is widespread among both free-living and par- 500 random single-copy orthologs from ATCC24927, the TWF154 reference asitic nematodes. It is the most abundantly excreted ascaroside of all plant- genome, and 18 additional wild isolate genomes. haptotyla (strain parasitic nematodes analyzed so far (60, 61) and it is the most abundant in CBS200) was used as the outgroup. The genomes of 18 wild isolates of A. several entomopathogenic nematodes (62). Ascaroside #18 was synthesized oligospora were assembled from 18 million 250-bp Illumina reads using AAFTF as reported (63) and was of greater than 98% purity. Micrographs were (68) and annotated using Funannotate (65). Further details about the as- taken after 24 h after the addition of nematodes or ascaroside using a Nikon sembly and annotation of the fungal isolates, as well as tree construction, SMZ 745T stereo microscope. For each plate, 3 images were randomly taken can be found in SI Appendix. within 0.5 cm from the edge of the plate using a 40× magnification, and the sum of traps in the 3 images was recorded. To estimate the survival rate of C. Identification and Deletion of the GPB1 Homolog in A. oligospora. The GPB1 elegans after exposure to A. oligospora, the number of moving nematodes homolog of A. oligospora was identified with Blast2GO 5 Pro (69) by per- was computed every 2 h for a total of 12 h using a stereo microscope. forming a local BLAST using the amino acid sequence of Gpb1 orthologs of Nematodes crawling on the wall of the plate, and therefore not in contact N. crassa (UniProt ID Q7RWT0), nidulans (Q5BH99), Fusarium with the fungal hyphae, were excluded. Survival rates are presented as the oxysporum (Q96VA6), and S. cerevisiae (A6ZP55) as the query and the pro- percentage of moving worms over the total number of worms. The time for teome of the T727 strain of A. oligospora as the database. GPB1 was deleted trap emergence after contact with nematodes was measured in the same by a homologous recombination-based strategy (70). Detailed methods in- conditions as described for the previous assays but 5-cm LNM plates, 3 d of cluding primers used for generating the gpb1 deletion mutant are described growth (25 °C, dark), and 300 adult N2 C. elegans were used. A Zeiss Stemi in SI Appendix. 305 microscope with a Zeiss Axiocam ERc 5s camera was used to automati- cally capture images of the colony edge every 60 s for a total of 12 h. Either 3 Data Availability. The genomic data presented in this paper have been de- × or 4 time-lapse videos were taken for each strain consisting of 2,560- posited to National Center for Biotechnology Information GenBank. The ac- × 1,920-pixel images representing an area of about 5.7 4.3 mm of the edge cession numbers are SOZJ00000000 (TWF154 reference genome), MN972613 of the fungal colony. The time-lapse videos were visually inspected to record (TWF154 mitochondria), WIQW00000000 (TWF102), WIQX00000000 (TWF103), the time point at which the first trap arose in the imaged areas. For the WIWS00000000 (TWF106), JAABOH000000000 (TWF128), JAABLO000000000 competition assay, a hygromycin B (HygB)-resistant TWF154 strain was (TWF132), JAAGKP000000000 (TWF173), WIPF00000000 (TWF191), WIPG00000000 constructed by transforming a HygB resistance cassette [amplified from (TWF192), JAABOI000000000 (TWF217), WIWR00000000 (TWF225), WIRA00000000 vector pAN7-1 (64)] into TWF154 protoplasts (SI Appendix). The HygB-resistant (TWF569), WIRB00000000 (TWF594), WIWT00000000 (TWF679), WIQZ00000000 TWF154 strain showed no defect in growth, conidiation, and trap morpho- (TWF703), WIQY00000000 (TWF706), WIWQ00000000 (TWF751), JAABOE000000000 genesis compared with WT. Single spores of both HygB-resistant TWF154 and (TWF788), and JAABOJ000000000 (TWF970). References to these accession TWF106 (HygB-sensitive) were collected and transferred together to the numbers can also be found throughout this paper and in SI Appendix. center of replicated 5-cm LNM plates. After 4 d of growth, 300 adult N2 C. elegans were added to half of the plates and all fungal colonies were ACKNOWLEDGMENTS. We thank John Wang, Jun-Yi Leu, and Sheng-Feng further grown for 3 more days to allow for trapping and digestion of the Shen for their helpful comments and suggestions about this work. We also nematodes by the fungi. A spore suspension was then collected from the thank Ling-Mei Hsu and A-Mei Yang for their technical assistance. This work plates by adding 2 mL ddH2O and scratching the surface of the fungal was supported by the start-up fund of Academia Sinica and Taiwan Ministry colony, followed by centrifugation (1 min, 13,200 rpm) and removal of the of Science and Technology 106-2311-B-001-039-MY3 (to Y.-P.H.).

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